Methods of alleviating symptoms of ocular surface discomfort using medical ice slurry

ABSTRACT

Disclosed herein is a method of alleviating symptoms of ocular surface discomfort, the method comprising: topically applying a cold slurry adjacent to a corneal limbus of an eye of a patient, wherein the cold slurry comprises water and a freezing point depressant, wherein the topical application of the cold slurry is configured to cause a degree of numbing of a cornea of the eye for a period of time, and wherein an ocular sensation of the eye is restored following the period of time.

This application is a national stage application of InternationalApplication No. PCT/US2021/024514, entitled “METHODS OF ALLEVIATINGSYMPTOMS OF OCULAR SURFACE DISCOMFORT USING MEDICAL ICE SLURRY,” filedon Mar. 26, 2021, which claims the benefit of and prior to U.S.Provisional Patent Application No. 63/000,922, filed on Mar. 27, 2020,the disclosures of each of which are hereby incorporated by reference intheir entireties.

TECHNICAL FIELD

The present invention relates generally to apparatuses, systems, andmethods for creating and administering a biomaterial such as a coldslurry. More particularly, the present invention relates to systems andmethods for treating ocular surface discomfort by administering a coldslurry to a subject to cause ocular hypesthesia in a safe and effectivemanner.

BACKGROUND

The cornea of the eye is a transparent, avascular tissue that measuresapproximately 11-12 mm horizontally and 9-11 mm vertically. Sridhar, M.S., Anatomy of cornea and ocular surface. Indian Journal ofOphthalmology, 66(2), 190-194 (February 2018). It is located on theoutermost surface of the eye, positioned in front of the pupil and iris,in order to refract light as it enters.

Innervation to the cornea begins at the brain stem, where a largesensory root branches off the pons and attaches to the trigeminalnucleus caudalis—located at the lateral portion of the medulla. Fromthere, the trigeminal nerve branches off into three divisions, one ofwhich is the ophthalmic division. This root divides into three morebranches, with one extension, called the nasociliary nerve. This purelysensory nerve travels along the superior portion of the orbital cavityand contributes smaller branches to the cornea. The two types ofdivisions from this nerve are called short ciliary nerves and longciliary nerves. The short ciliary nerves pass through a sensory root,into the ciliary ganglion, then exit that nucleus to pierce the scleraand enter the peri-choroidal space, where they can move into the cornea.Belmonte, C., Tervo, T. T., & Gallar, J. (2011). CHAPTER 16—SensoryInnervation of the Eye. Adler's Physiology of the Eye (Eleventh Edition,pp. 363-384). Elsevier Inc.

The peri-choroidal space is located between the sclera, the outermostlayer of the eyeball, and the choroid, the heavily vasculated layer thatis responsible for providing nutrients to the structures of the eye.There are about 8-10 short ciliary nerves that puncture the sclera, butthese nerves branch into about 15-20 divisions once inside theperi-choroidal space. In regard to the long ciliary nerves, there areover 50 branches that pierce the sclera and divide again inside theperi-choroidal space. At the limbus, the junction between the sclera andcornea, the nerves lose their myelin sheath and continue as free nerveendings. The nerves collect sensory signals from the cornea and sendthem backwards, towards the brain stem. Belmonte, C., Tervo, T. T., &Gallar, J. (2011). CHAPTER 16—Sensory Innervation of the Eye. Adler'sPhysiology of the Eye (Eleventh Edition, pp. 363-384). Elsevier Inc. Notall the details of corneal innervation are entirely well-understood andmay vary somewhat patient to patient. There may be some contributionfrom other nerve fibers or some normal anatomical variations in themutes of innervation.

The free nerve endings are located under the corneal epithelium, theanterior layer protecting the corneal structure, and often contribute topainful ocular sensations. When patients are bothered by these symptoms,the condition is termed dry eye syndrome (DES), also known as ocularsurface disease (OSD). Causes of this condition are multifactorial. Oneimportant cause is inadequate amounts of aqueous tear production,causing the eye to lack hydration and lubrication. Other causes ofocular surface disease can include meibomian gland dysfunction or damageto the corneal epithelium.

These “dry eye-induced alterations to the properties of corneal afferentneurons and the central processing of corneal input may have significantconsequences for both the regulation of tearing and ocular pain.”Memonnies, C. W., The potential role of neuropathic mechanisms in dryeye syndromes, Journal of Optometry, 10, 5-13 (2017). Importantly, somepatients continue to have ocular surface pain even though their ocularsurface returns to a clinically normal appearance. This situationpresents a clinical challenge because the cause is thought to besomatosensory dysfunction of the corneal innervation that persists longafter the original insult that excited the nerves.

Other sources of corneal discomfort can include postoperative pain,potentially after photorefractive keratectomy, which is a procedure usedto treat refractive error that requires removal of the cornealepithelium before applying excimer laser ablation. Other surgicalprocedures can also lead to corneal discomfort, including proceduresthat do not necessarily involve removal of the epithelium, but where theepithelium undergoes a mild to moderate desiccation during theprocedure. A patient can also experience ocular discomfort followingocular trauma (e g., corneal abrasions) and laser in-situ keratomileusis(LASIK) surgery.

There are three different types of nociceptive receptors innervating thecornea. Twenty percent of corneal nociceptors are Aδ mechanoreceptors,which are responsible for fast-conducting sharp, painful stimuli, causedby aggravation to the ocular surface. Seventy percent of cornealnociceptors are polymodal, which are stimulated by corneal nerve damageand cause neuropathic pain and “reflexive tearing.” Levitt, A. E., etal., Chronic dry eye symptoms after LASIK: parallels and lessons to belearned from other persistent post operative pain disorders, MolecularPain, 11:21 (2015). The last ten percent of corneal nociceptors areC-fiber cold receptors, which play a crucial role in maintaining basaltear secretion. These receptors are highly sensitive to temperaturechange within corneal tissue, and LARK surgery can cause evaporation oftears on the tear film surface, lowering the temperature by about 0.3degrees per second, thus influencing C-fiber signals. (Levitt et al.,2015).

Many mechanisms, including dryness, prior surgery, dysfunction of theeyelid glands, or prior chemical irritation, may lead to the clinicalsyndrome of OSD, notable for signs of ocular irritation and symptomscharacterized as dryness, burning, or discomfort. Even after the initialinsult for the mechanism has resolved, i.e. normal lubrication of theeye has been restored, patients may still report significant symptoms ofocular surface discomfort, despite their ocular surface having onlyminimal signs of disease, suggesting a component of hypersensitizationor allodynia. Indeed, literature references note “the status of theocular surface alone is not sufficient to understand dry eye and thatcorneal somatosensory function . . . must be considered when evaluatinga patient with dry eye.” Spierer O, Felix E R, McClel-lan A L, et al.Corneal mechanical thresholds negatively associate with dry eye andocular pain symptoms. Invest Ophthalmol Vis Sci. 57:617-625 (2016). Thissituation presents a quandary for the treating physician—the patient hasresidual pain and discomfort with a normal appearing ocular surface(corneal somatosensory dysfunction). Additional lubrication and othertherapies targeted to improve the ocular surface are, as expected, nolonger of any help to these patients.

Current treatment methods for pain associated with dry eyesyndrome/ocular surface disease, PRK, or LASIK surgery, or cornealsomatosensory dysfunction are either limited, of transient value, orassociated with negative side effects. Dry eye syndrome is most commonlytreated with warm compresses, over-the-counter artificial tears, orprescription eye drops targeting improved tear production or reducinginflammation. Physicians may also recommend topical ocular lubricants,hygiene products that clear debris from underneath the eyelids. Thesemethods work by softening meibum, the oily, lipid-rich secretion frommeibomian glands, in order to help spread tear production over thecornea. The limitation to these therapies is the short-term relief andneed for continuous application. Lubricants or artificial tears maysoothe irritation, but do not actually address the cause of dry eye andmay also contribute to increased debris collecting under the eyelid.Shen Lee, B., et al., Managing dry eye disease and facilitatingrealistic patient expectations: A review and appraisal of currenttherapies, Clinical Ophthalmology, 14 119-126 (January 2020).

Regarding postoperative pain management for photorefractive keratectomyand LASIK eye surgery, topical NSAIDs and soft bandage contact lensesare the most common treatment. NSAID medications prevent the productionof prostaglandin, a hormone-like substance involved in inflammation,that occurs with corneal tissue damage. Pathak, A. K., & Karacal, H.,(2019). Pain reduction after photoablation. EyeWiki by the AmericanAcademy of Ophthalmology. Topical NSAIDs carry a risk of corneal damage,such as erosions, defects, delayed corneal epithelial healing, orcorneal melting (which can lead to vision loss). With soft bandagecontact lenses, this method can stimulate the re-growth of epithelialcells, and act as a delivery system for antibiotics or topical NSAIDs.However, bandage contact lenses can promote bacterial growth and areoften not be efficient in relieving pain. Shetty, R., et al., Painmanagement after photorefractive keratectomy, Journal of CataractRefract Surgery, 45(7):972-976 (2019).

Acute ocular pain can also be treated with topical ophthalmic anestheticdrops, such as Proparacaine Hydrochloride and Tetracaine Hydrochloride.These aqueous solutions are given as short-term treatment for pain, orwhen measuring intra-ocular pressure, removing foreign bodies, soothingsutures in the cornea, or as a preoperative anesthetic for ophthalmicsurgery. Topical anesthetics can block conical nerves from sendingpainful stimuli for about 15-20 minutes per dose. With this short-termpain relief, patients require continuous application, but chronic usecan eventually lead to conical toxicity. Toxic effects on the corneainclude damaging stromal keratocytes, which are cells that play asignificant role in healing trauma to the cornea. If epithelial cellscannot migrate across the cornea, the epithelium will eventually startto slough off and lead to chronic non-healing of corneal epithelium.

Maintaining some perception of pain, however, is critical to the normalfunction of the healthy cornea. Neuropathic keratopathy, also known asneurotrophic keratitis, is a syndrome where due to a pathological lackof sensation of the corneal and conjunctiva, the ocular surfaceexperiences a syndrome that progresses from tear film abnormalities, toepitheliopathy, to eventual stromal lysis. For true neuropathickeratopathy, an eye must have a lack of sensation of the cornea andconjunctiva due to pathological destruction to the trigeminal nerve,which can occur from surgery intended to treat trigeminal neuralgias,surgery of acoustic neuromata, or from infections such as herpes zosterophthalmicus or leprosy. Other forms of neuropathic keratopathy occurfrom the misuse of topical anesthetics. In rabbit models, typicaltrophic changes in the corneal epithelium have been shown aftercontrolled thermocoagulation of the trigeminal ganglion in rabbits. Thisdenervation was found to markedly affect the proliferative activity ofthe epithelium, and mitosis was sparse.

As explained above, the cornea is exquisitely sensitive to pain orperturbation. There are a host of human clinical conditions that causemild to severe corneal pain and discomfort, all of which couldpotentially be addressed by the development of a safe and efficacioustreatment for corneal pain. The current state of topical numbing dropsonly numb the cornea for a matter of minutes and chronic use isassociated with severe morbidities including conical infections andcorneal melting. Further, traditional approaches for treating ocularpain result in complete anesthesia of sensation to the eye, which can bevery problematic in the chronic context due to the risk of developmentof neuropathic keratopathy. Furthermore, in eyes that are chronicallyinflamed and painful, a corneal somatosensory dysfunction becomes thepredominant feature of the pain syndrome. In summary, there are manypatients with debilitating ocular surface discomfort that can beassociated with active conical pathology or can persist long after theoriginal injury with no detectable ongoing pathology. Clearly there is alarge unmet clinical need for development of a longer-acting safeconical anesthetic therapy that partially blocks conical sensation andsignificantly decreases patient discomfort.

SUMMARY

In one aspect, the invention provides for a method of alleviatingsymptoms of ocular surface discomfort, the method comprising: topicallyapplying a cold slurry adjacent to a corneal limbus of an eye of apatient, wherein the cold slurry comprises water and a freezing pointdepressant, wherein the topical application of the cold slurry isconfigured to cause a degree of numbing of a cornea of the eye for aperiod of time, and wherein an ocular sensation of the eye is restoredfollowing the period of time.

In some embodiments, the cold slurry is applied posterior to the corneallimbus.

In some embodiments, the period of time is more than about 2 dayswithout topically applying the cold slurry an additional time on any dayfollowing the first day of topical application.

In some embodiments, the period of time is more than about 7 dayswithout topically applying the cold slurry an additional time on any dayfollowing the first day of topical application.

In some embodiments, the freezing point depressant is glycerol.

In some embodiments, the ocular sensation of the eye is restored afterabout 21 days following the topical application of the cold slurry.

In some embodiments, a sclera of the eye of the patient is cooled to atemperature of between about −6° C. and about 4° C. during the topicalapplication of the cold slurry.

In some embodiments, the cold slurry is topically applied for betweenabout 5 minutes and about 15 minutes.

In some embodiments, an additional amount of the cold slurry istopically re applied at about every 90 seconds.

In some embodiments, the method further comprises placing a contact lenson the eye of the patient prior to topically applying the cold slurry.

In some embodiments, the cold slurry is configured to be a pasteconsistency.

In another aspect, the invention provides for a method of alleviatingsymptoms of ocular surface discomfort, the method comprising: placing aprotective covering over a cornea of an eye of a patient; and topicallyapplying a cold slum to a bulbar conjunctiva of the eye of the patient,wherein the topical application of the cold slurry causes a prolongedreduction of pain of the eye of the patient, and wherein a partialsensation of the cornea of the eye of the patient is maintained duringthe prolonged reduction of pain.

In some embodiments, the cold slurry is applied posterior to the corneallimbus.

In some embodiments, the cold slurry is applied over the protectivecovering.

In some embodiments, the prolonged reduction of pain lasts more thanabout 7 days without topically applying the cold slurry an additionaltime on any day following the first day of topical application.

In some embodiments, the prolonged reduction of pain lasts more thanabout 2 days without topically applying the cold slurry an additionaltime on any day following the first day of topical application.

In some embodiments, the prolonged reduction of pain lasts more thanabout 14 days without topically applying the cold slurry an additionaltime on any day following the first day of topical application.

In some embodiments, the symptoms are due to dry eye syndrome or cornealsomatosensory dysfunction.

In some embodiments, a sclera of the eye of the patient is cooled to atemperature of between about −6° C. and about 4° C. during the topicalapplication of the cold slurry.

In some embodiments, the protective covering is a contact lens, andwherein the contact lens prevents the cornea of the eye from freezing.

In another aspect, the invention provides for a method of alleviatingsymptoms of ocular surface discomfort, the method comprising:administering a cold slurry to an eye of a patient, wherein the coldslurry comprises water and a percentage of ice particles, wherein theadministration of the cold slurry causes a prolonged hypesthesia of theeye, wherein an ocular sensation of the eye is restored following theprolonged hypesthesia, and wherein the administration of the cold slurrydoes not cause permanent damage to a cornea of the eye.

In some embodiments, the method further comprises treating a conditionselected from the group consisting of dry eye syndrome, chronic eyepain, post-operative pain, post-photorefractive keratectomy pain,post-LASIK pain, post-cataract surgery pain, and post open globe injuryrepair pain, post-corneal injury, corneal somatosensory dysfunction,allodynia, and pain from acute injury.

In some embodiments, the cold slurry is administered via injection.

In some embodiments, the cold slurry is injected into a subconjunctivalspace.

In some embodiments, the cold slurry is administered via topicalapplication.

In some embodiments, the percentage of ice particles is between about20% and 40%.

In some embodiments, the temperature of the cold slurry is between about−20° C. and about −5° C.

In another aspect, the invention provides for a method of alleviatingsymptoms of ocular surface discomfort, the method comprising: topicallyapplying a cold slurry on or proximal to an ocular surface of an eye ofa patient, wherein the topical application of the cold slurry causes aprolonged hypesthesia of a cornea of the eye, wherein an ocularsensation of the eye is restored following the prolonged hypesthesia,and wherein the topical application of the cold slurry does not causepermanent damage to the cornea of the eye.

In some embodiments, the cold slurry is applied proximal to the corneallimbus.

In some embodiments, the prolonged hypesthesia lasts more than about 1day following a single treatment of the topical application of the coldslurry.

In some embodiments, the ocular sensation of the eye is restored byabout 30 days following the topical application of the cold slurry.

In some embodiments, the cold slurry is topically applied for betweenabout 5 minutes and about 15 minutes.

In some embodiments, the method further comprises placing a contact lenson the rye of the patient prior to topically applying the cold slurry,and wherein the contact lens prevents the cornea of the eye fromfreezing.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures depict illustrative embodiments of the invention.

FIG. 1 depicts a freezing point depression graph for liquid water, asolution containing 10% glycerin volume by volume (v/v), and a solutioncontaining 20% glycerin (v/v).

FIG. 2 is a table showing the breakdown by volume and weight ofcomponents of an exemplary biomaterial that can form an injectable coldslurry.

FIG. 3 is a graph showing the characterization of ice content of coldslurries having crystallization set points of −5.5° C. and −8.1° C.

FIG. 4 shows diagrams of a human eye showing different areas of the eye(4A) and degrees for anatomical reference (4B).

FIG. 5 is a graph showing real-time scleral temperature monitoring inrabbits following administration to the eye of a topically applied coldslurry (solid line) and an injected cold slurry (dashed line).

FIG. 6 is a graph showing hypesthesia in rabbits' eyes over timefollowing administration to the eye, with the cornea exposed, of aninjected cold slurry (diamond), a topically applied cold slurry(triangle), and a topically applied slurry at room temperature (square).

FIG. 7 is a graph showing hypesthesia in rabbits' eyes over timefollowing administration to the eye, with the cornea not exposed, of atopically applied cold slurry.

FIG. 8 are images of fluorescein stained rabbit cornea showing cornealhealing over time following intentional 8 mm corneal abrasion (8A) as acontrol group and following topical application of cold slurry (8B).

FIG. 9 is a graph showing hypesthesia in 6 rabbits' eyes over time aftera combination treatment in which a cold slurry was topically appliedfirst and then injected. In three rabbits (shown with a diamond, square,and triangle), the injected slurry did not contain liposomes, while inthe other three rabbits (shown with an “X”, star, and circle), theinjected slurry contained liposomes.

DETAILED DESCRIPTION

The present disclosure is drawn to apparatuses, devices, systems, andmethods of treating ocular surface discomfort with a biologicalmaterial, such as a cold slurry. In some embodiments, the biomaterial isa cold slurry (e.g., ice slurry) that can be delivered via topicalapplication or via injection into the eye of a human patient or asubject (e.g., a human who is not a patient or a non-human animal) forprophylactic or therapeutic purposes to reduce ocular discomfort. Thesystems and methods disclosed herein provide for a hypesthesia of theeye that is unexpectedly long-lasting. The hypesthesia may cause along-lasting corneal numbing followed by a restoration of ocularsensation within days or weeks following application of cold slurrytreatment, without causing permanent damage to the cornea or disruptingthe progress of corneal healing.

In some embodiments, the cold slurry can be applied topically to achievea desired therapeutic effect such as amelioration or treatment of ocularsurface discomfort through long-term corneal numbing. In someembodiments, the therapeutically effective cold slurry is comprisedentirely of water and excipient materials (i.e., materials without anactive pharmaceutical compound). In other embodiments, the cold slurryfurther comprises a known active pharmaceutical compound. In someembodiments, a layer of protection, like a contact lens, is applied tothe cornea prior to the topical application of the slurry. In someembodiments, the eyelid is protected from topical application of theslurry by inserting a plastic, or other non-thermally conductivematerial, speculum into a subject's eye.

In some embodiments the length of time that the slurry is applied to asubject's eye can be varied to induce a greater or milder hypesthesia.In some embodiments, the temperature of the slurry applied or injectedinto a subject's eye is varied to induce a greater or milderhypesthesia. In some embodiments, the hypesthesia decreases over time tothe point where it is not noticeable. In other embodiments, where asubject's eye may be particularly sensitive, a greater hypesthesia isinduced to numb more of the nerves in the subject's eye.

In some embodiments, a container (e.g., vial, syringe) containing abiomaterial is received at a clinical point of care. The biomaterial maybe received in a crystallized (or partially crystallized) state. In someembodiments, the final product to be administered via topicalapplication or injection to a human patient or a subject (such as ahuman who is not a patient or a non-human animal) is a cold slurrycomprised of sterile ice particles of water and varying amounts ofexcipients or additives such as freezing point depressants. For example,the percentage of ice particles in the cold slurry can constitute lessthan about 10% by weight of the slurry, between about 10% by weight andabout 20% by weight, between about 20% by weight and about 30% byweight, between about 30% by weight and about 40% by weight, betweenabout 40% by weight and about 60% by weight, more than about 60% byweight, and the like. The sizes of the ice particles will be controlledto allow for flowability through a vessel of various sizes (e.g. needlegauge size of between about 7 and about 43) as described in U.S.application Ser. No. 15/505,042 (Publication No. US2017/0274011) andincorporated herein by reference. Further, other methods may be used tocondition the size of the ice particles to allow for flowability througha vessel of various sizes. In some embodiments, the majority of iceparticles have a diameter that is less than about half of the internaldiameter of the lumen or vessel used for injection. For example, iceparticles can be about 1.5 mm or less in diameter for use with a 3 mmcatheter.

There are a variety of techniques that may be used to prepare a coldslurry. This disclosure is not limited to any particular method ortechnique.

In some embodiments, one or more excipients may be included in the coldslurry. An excipient is any substance, not itself a therapeutic agent,used as a diluent, adjuvant, and/or vehicle for delivery of atherapeutic agent to a subject or patient, and/or a substance added to acomposition to improve its handling, stability, or storage properties.Excipients can constitute less than about 10% volume by volume (v/v) ofthe cold slurry, between about 10% v/v and about 20% v/v of the slurry,between about 20% v/v and about 30% v/v, between about 30% v/v and 40%v/v, and greater than about 40% v/v. Various added excipients can beused to alter the phase change temperature of the cold slurry (e.g.,reduce the freezing point), alter the ice percentage of the cold slurry,alter the viscosity of the cold slurry, prevent agglomeration of the iceparticles, prevent dendritic ice formation (i.e., crystals withmulti-branching “tree-like” formations, such as those seen insnowflakes), keep ice particles separated, increase thermal conductivityof fluid phase, or improve the overall prophylactic, therapeutic, oraesthetic efficacy of the cold slurry.

One or more freezing point depressants can be added as excipients toform cold slurries with freezing points below 0° C. Depressing thefreezing point of the slurry allows it to maintain flowability andremain injectable while still containing an effective percentage of iceparticles. Suitable freezing point depressants include salts (e.g.,sodium chloride, betadex sulfobutyl ether sodium), ions, LactatedRinger's solution, sugars (e.g., glucose, sorbitol, mannitol,hetastarch, sucrose, (2-Hydroxypropyl)-β-cyclodextrin, or a combinationthereof), biocompatible surfactants such as glycerol (also known asglycerin or glycerine), other polyols (e.g., polyvinyl alcohol,polyethylene glycol 300, polyethylene glycol 400, propylene glycol),other sugar alcohols, or urea, and the like. Other exemplary freezingpoint depressants are disclosed in U.S. application Ser. No. 15/505,042(Publication No. US2017/0274011) and are incorporated in their entiretyherein. In other embodiments, a slurry paste is formed that has theconsistency of toothpaste and has a consistency that is optimal fortopical application.

The concentrations of freezing point depressants will determine the iceparticle percentage of the cold slurry and its flowability andinjectability. The degree of freezing point depression can be calculatedusing the following formula as described in U.S. application Ser. No.15/505,042 (Publication No. US2017/0274011), incorporated herein:ΔT _(F) =K _(F) biwherein ΔT_(F) is the freezing point depression (as defined byT_(F (pure solvent))−T_(F (solution))), K_(F) is the cryoscopicconstant, b is molality, and i is the van't Hoff factor representing thenumber of ion particles per individual molecule of solute. Other methodsof computing freezing point depression can also be used, as disclosed inU.S. application Ser. No. 15/505,042 (Publication No. US2017/0274011).

Referring to FIG. 1, a freezing point depression graph is shown for purewater T1, a mixture of water and 10% (v/v) glycerin T2, and a mixture ofwater and 20% (v/v) glycerin T3. In this graph, all the substances wereplaced in a freezer having a constant temperature of −20° C. Thetemperature was measured using a thermometer placed in each substance.The graph shows that a mixture of water and glycerin will have adifferent freezing point than that of pure water, which means thesolution can be cooled to below 0° C. and only be partiallycrystallized. The graph shows that cooling causes pure water T1 tocrystallize at an equilibrium freezing point of 0° C. This is indicatedby the period of time where the pure water remains at a temperature ofabout 0° C., from about 1.3 hours to about 4.4 hours, which beginsimmediately after pure water T1 passes a supercooling point at about −6°C. Having an equilibrium window of crystallization (i.e., the “flatline” portion of pure water T1 in FIG. 1) is typical for a pure solvent.For the 10% glycerin solution T2, cooling causes the solution to begincrystallizing at an initial freezing point of about −3° C. after about2.2 hours, and the crystallization continues as the temperature of thesolution drops further to about −8° C. after about 6 hours. The initialcrystallization occurs immediately after 10% glycerin solution T2 passesa supercooling point at about −8° C. (which can vary from sample tosample, e.g., supercooling point of between about −15° C. and about −3°C.), shown at around 2.2 hours. Having a descending temperature windowof crystallization for the 10% glycerin solution T2 is typical for asolution (i.e., impure mixture). Similarly, for the 20% glycerinsolution T3, cooling causes the solution to begin crystallizing at aninitial freezing point of about −7° C. after about 3.5 hours (followingan initial supercooling point which can vary from sample to sample,e.g., between about −25° C. and about −5° C.), and the crystallizationcontinues as the temperature of the solution drops further to about −11°C. after about 6 hours and continues to decline thereafter past 6.5hours. The initial crystallization occurs immediately after 20% glycerinsolution T3 passes a supercooling point at about −14° C., shown ataround 3.5 hours. Similar to the trace for 10% glycerin solution T2, thedescending temperature window of crystallization for 20% glycerinsolution T3 is typical for a solution.

Referring to FIG. 2, this chart shows the components of an exemplarybiomaterial that can form a cold slum. This chart shows that thepercentage of ice for an exemplary biomaterial can be calculated for aparticular temperature. The exemplary slurry contains 30% ice by mass(weight by weight; w/w) at −10° C. This exemplary slurry has 80 mL ofsaline (0.9% NaCl) and 20 mL of glycerol (i.e., glycerin). In weight,such a slurry has about 79.6 g of pure water, about 0.72 g of sodiumchloride, and about 25.2 g of glycerol (approximately 20% v/v). In otherembodiments, the slurry could contain higher or lower percentages ofglycerol by adjusting the relative volume of glycerol to saline. Forexample, other suitable slurries contain about 10% glycerol (v/v),between about 10% and about 20%, glycerol, about 30% glycerol, or morethan about 30% glycerol. If an active pharmaceutical compound is to beadded to the slurry, the concentration of saline can be adjustedaccordingly to maintain the desired concentration of excipients such asglycerol. The percentage of ice will vary depending on the compositionof the biomaterial.

Referring to FIG. 3, different slurry compositions (batches) arecharacterized with respect to their temperature profiles and icecontent. The different slurry batches were placed into a copper platethat is heated to 40° C. and has thermocouple wires that measure changesin temperature of the slurry over time. The plotted data showstemperature change over time for three different slurry batches. Thetemperatures are measured at two different positions for each slurry:embedded inside of the copper plate (traces A_(C), B_(C), and C_(C)) andin the middle of the copper plate exposed to the outside of the plate(traces A_(M), B_(M), and C_(M)). The temperature traces show threeseparately created slurry batches: a slurry composition having 15%glycerin (having a temperature setpoint of −8.1° C.) is represented bytraces A_(C) and A_(M), and two different slurry batches both having 10%glycerin (having a temperature setpoint of −5.5° C.) are represented bytraces B_(C) and B_(M), as well as traces C_(C) and C_(M). When a slurrybatch is first introduced into the copper plate, the thermocouple wireembedded inside the plate (traces A_(C), B_(C), and C_(C)) initiallymeasures the warm temperature of the heated plate (e.g., 31° C. fortrace A_(C) at timepoint 0) and then reaches an equilibrium at a lowertemperature due to the cooling effect of the introduced slurry (e.g.,22° C. for trace A_(C) at around 2 minutes). On the other hand, for thethermocouple wire located in the middle of the plate, when a slurry isfirst introduced into the copper plate it immediately contacts thethermocouple wire since that wire is exposed. This causes an initiallynegative temperature reading in the middle position due to thecrystallized slurry contacting the wire (e.g., −5° C. for trace A_(M) attimepoint 0) followed by an equilibrium at a warmer temperature as theslurry begins to melt on the heated plate (e.g., 18° C. for trace A_(M)at around 4 minutes). The thermocouple wire exposed to the outside ofthe plate (traces A_(M), B_(M), and C_(M)) can be used to detect phasetransitions during which the crystallized slurry begins to melt. Thegraph shows that the two slum compositions with 10% glycerin reach theirphase transition at similar timepoints (at around 4 minutes for traceB_(M), and at around 2.7 minutes for trace C_(M)), which differ from thephase transition for the 15% glycerin slurry (phase transition occurs ataround 0.2 minutes for trace A_(M)). The graph also shows that the twoslurry batches having the same composition (10% glycerin: traces B_(C)and B_(M) and traces C_(C) and C_(M)) reach equilibrium (as measured bythe two thermocouple wire positions) in a similar time frame and atsimilar temperatures of between about 15° C. and 19° C. depending on thelocation of the thermocouple (middle/bottom). On the other hand, theslurry with a different composition (15% glycerin: traces A_(C) andA_(M)) has a different temperature profile from the other two, reachingan equilibrium sooner at the temperature of between about 19° C. and 22°C. depending on the location of the thermocouple (middle/bottom). FIG. 3therefore demonstrates that slurries of different compositions havedifferent temperature profiles and batch to batch consistency existsacross slurries having the same composition (e.g., the slurryrepresented by B_(C) and B_(M) and slurry represented by C_(C) and C_(M)have similar temperature profiles which is different from that of slurryrepresented by A_(C) and A_(M)).

With reference to FIG. 4A, a diagram of an eye is shown depicting thesclera zone 2, sclera zone 3, cornea 1, and the corneal limbus (dottedline between cornea 1 and sclera zone 2. FIG. 4A is reproduced fromAndreoli C M, Gardiner M F. Open globe injuries: Emergent evaluation andinitial management. In: UpToDate, Post TW (Ed), UpToDate, Waltham, Mass.FIG. 4B shows a diagram of an eye with a superimposed protractor showingangles in degrees (°) with respect to the eye. In this diagram, 90°represents the superior most position along the eye.

In some embodiments, the cold slurries described herein can be appliedtopically, or alternatively injected, to achieve long-lastinghypesthesia that reduces ocular surface discomfort. Hypesthesia refersto a reduction of ocular discomfort or pain without a complete blockageof ocular sensation. Hypesthesia is therefore distinguished fromanesthesia, anesthesia being characterized as a more profound blockageof ocular sensation. Hypesthesia may include corneal numbing whichcauses a reduction in pain response, while maintaining an otherwisenormal functioning of the eye, including a normal healing process.Anesthesia, on the other hand, may lead to abnormal functioning of theeye since all corneal sensation is lost. Corneal sensation is importantfor normal functioning of the eye including the initiation of protectivemechanisms such as blinking and tear production.

One approach is to apply drops of the cold slurry to the ocular surfacewhich may vary in volume from 1 to 100 microliters, preferably fromapproximately 10 to 80 microliters. As drops, the formulation may bedirectly administered to the ocular surface. Alternatively, the corneamay be scraped first, and drops may be subsequently administered. Insome embodiments, the topically applied cold slurry has a more flowablepaste consistency, and a large quantity, could be applied topically(3-50 ml) to the ocular surface as a treatment.

In some embodiments, the cold slurries as described herein are appliedtopically posterior to the corneal limbus, e.g., the area denoted assclera zone 2 in FIG. 4A, for between about 1 minute and about 20minutes. In some embodiments, the cold slurry is applied for betweenabout 5 minutes and about 10 minutes. In some embodiments, cold slurryis administered every 1 to 10 seconds for 1 to 20 minutes into each eye.This treatment could be repeated several times over a short period oftime (e.g., 5-20 min). In some embodiments, the cold slurry is appliedtopically posterior to the corneal limbus for about 10 minutes, withfresh slurry re-applied every 90 seconds until 10 minutes is reached.

In some embodiments, during the topical application, sensitive ocularstructures are protected from encountering the cold slurry in order tolimit potential side effects. Protection of the corneal surface maylimit some or all corneal cell damage or refractive changes caused byfreezing the corneal tissue. Protection of the palpebral conjunctiva andeye lids may prevent redness, swelling and inflammation that would notbe related to the treatment effect. By selectively applying ice to theocular surface posterior to the limbus over the bulbar conjunctiva(Corresponding to the anterior anatomical area known as zone 2, FIG.4A), potential adverse effects to the cornea may be minimized.

In some embodiments, a protective contact lens or other protective covercan be applied to sit on the cornea to protect the cornea from damage.In some embodiments, a corneal covering completely blocks the coldslurry from contacting the cornea surface directly. In some embodiments,a lid speculum is used to keep the eye lids open during cold slurrytopical application. In some embodiments, the speculum is made of athermally non conductive material such as plastic or another othernon-conductive material known in the art. A thermally non-conductivematerial may be used for the speculum to prevent the eyelids (inside andoutside) from freezing, which could cause damage to the eyelids, duringcold slurry treatment. In some embodiments, the cold slurry is appliedto the sclera only and the cornea is protected from freezing. In someembodiments, preventing the cornea from freezing will ensure fasterhealing of the cornea.

A device could be used to control the areas exposed to the cold slurryto just the bulbar conjunctiva/sclera so that the cold slurry could notphysically contact or freeze adjacent tissues that would not relevant tothe desired clinical effect. In some embodiments, the cold slurryformulation does not have direct contact with the ocular surface at all.The cold slurry formulation may be contained within a material that isthermally conductive, e.g., a small metal or polymeric donut or otherprotective ring, thus providing a barrier against direct contact of theformulation with the ocular surface but allowing the requisite coolingto occur. In some embodiments, the device directs cooling only to theareas of potential therapeutic effect and prevents the device/coolingfrom contacting and affecting adjacent tissues in order to minimizeundesired side effects (e.g., potential irritation of the eye from theapplication of a hyperosmolar solution directly to the eye).

In some embodiments, the cold slurries described herein are injected asa subconjunctival bolus, at about every 2 minutes. Each injection couldprovide approximately 0.5-1.5 ml of frozen slurry and be repeated everyapproximately 2 minutes for the duration of desired treatment, or about10 minutes in total. In some embodiments, the cold slurry is injectedmom, directly near the axons of the ciliary nerves. The ciliary nervesare located at approximately 0° and 180° of the eye (FIG. 4B).

In some embodiments, a standard syringe is used to inject slurry.Alternatively, a syringe that has conditioned the slurry for injectionmay be used. In some embodiments, the syringe may have a needle of about18 G to about 25 G.

In some embodiments, real-time temperature sensing on the surface of theeye is performed during the treatment (e.g., cold slurry injection ortopical application). In some embodiments, the cold slurry is applied tocool the tissue (e.g., corneal surface, conjunctiva, or any other partof the eye) to less than about 0° C., less than about −1° C., less thanabout −2° C., less than about −3° C., less than about −4° C., or lessthan about −5° C. In some embodiments, the cold slurry is applied forover about one minute, preferably for between about 2 minutes and about10 minutes. The temperature of the cooled tissue and the length of timethat the slurry is applied can be varied to vary the hypesthesiaexperienced by a subject.

In some embodiments, the cold slurry is periodically readministered to asubject's eye over time to maintain therapeutic effects. There is arange of possible frequencies for topical administration and/orinjection. For example, treatment could be administered any one of thefollowing: once every other week; once a month; once every other month;once every third month, etc.

In some embodiments, cold slurry is used as a safe corneal numbingtreatment to treat corneal discomfort or pain. Various formulations ofcold slurries can be used with the methods described herein, includingthose described above. Additional specific embodiments of cold slurriesare described with reference to FIGS. 5-9 “ECT-4143” is a slurryformulation that comprises 15% glycerol, 30% L-α-phosphatidylcholineliposomes, and 0.9% saline (or phosphate buffered saline). In someembodiments, ECT-4143 is administered to the eye (topically or viainjection) at a temperature of between about −25° C. and −10° C.(temperature of the slurry). In some embodiments, ECT-4143 isadministered to the eye (topically or via injection) at a temperature ofabout −18° C. (temperature of the slurry, such as in the embodimentsdescribed below with reference to FIGS. 5-9). ECT-4143 is administeredevery approximately 90 seconds, with approximately 2-3 ml perapplication, until a total treatment time of 10 minutes is reached.

“ECT-1719” is a slurry formulation that comprises 15% glycerol and 0.9%saline (or phosphate buffered saline). In some embodiments. ECT-1719 isadministered to the eye (topically or via injection) at a temperature ofbetween about −20° C. and −5° C., or between about −15° C. and about−10° C. (temperature of the slurry). In some embodiments, ECT-1719 isadministered to the eye (topically or via injection) at a temperature ofabout −11° C. (temperature of the slurry, such as in the embodimentsdescribed below with reference to FIGS. 5-9). In some embodiments,ECT-1719 is injected in 0.7 ml volume per injection, with four totalinjections making up 2.8 ml total injected volume. ECT-1719 isadministered every approximately 120 seconds until a total treatment of10 minutes is reached

Referring to FIG. 5, real-time scleral temperature monitoring wasperformed in rabbits following administration to the eye of a topicallyapplied cold slurry (ECT-4143, solid line) in one rabbit and an injectedcold slurry (ECT-1719, dashed line) in a second rabbit. The temperaturemonitoring is achieved by cannulating the subtenon's space with a 25 gneedle containing a temperature probe at its distal end. As can be seenin FIG. 5, the scleral temperature of the rabbit that received theinjected cold slurry (ECT-1719) fluctuated between about 0° C. and about8° C. throughout the duration of the procedure (from about 0 secondsfollowing cold slurry injection to about 463 seconds following coldslurry injection). The sharp line in the graph at about 463 secondsrepresents conclusion of the study after 7.5 minutes and removal of thetemperature probe from the ocular tissue. The scleral temperature of therabbit that received the topically applied cold slurry (ECT-4143) waslower than for the injected cold slurry with fluctuations between about−6° C. and about 4° C. for the majority of the time during whichtemperature was recoded (between about 0 seconds following topicalapplication and about 600 seconds following topical application).Following topical application, the temperature of the sclera continuedto drop from about 4° C. at the time of application (about 0 seconds inFIG. 5) to about 0° C. after about 120 seconds. Following the initialperiod of scleral cooling, the temperature remained relatively stable atbetween about 0° C. and −5° C. from about 120 seconds after topicalapplication to about 520 seconds after topical application.Additionally, for a duration of between about 220 seconds and about 520seconds following topical application, the scleral temperature showedvery little variability, remaining stable at about −2° C. to about −3°C. After about 620 seconds following topical application, the treatmentis concluded and the temperature probe is removed, showing a sharpincrease in measured temperature as shown in FIG. 5.

The hypesthetic effect following cold slurry treatment is measured as aresponse to tactile stimulation of the eye using amonofilament/esthesiometer. Starting at 6 cm filament length anddecreasing by 0.5 cm increments, the eye is probed three times at eachlength until a blink response is elicited. The filament gets stiffer asit is shortened, therefore imparting more pressure on the eye whenprobed into the eye. The hypesthesia for each time point is based on agiven length of the monofilament. At each time point, the specificmonofilament length that is recorded is the shortest length (highestpressure) at which blink response is not present. For example, startingwith the longest monofilament of 6 cm, if the rabbit does not blink whenprobed, the next monofilament of 5.5 cm length is used to probe the eye.If the rabbit does not blink again, the next monofilament length of 5 cmis used. Now, if the rabbit does blink, the previous length of 5.5 cm isrecorded because this is the shortest length at which there was no blinkresponse (reflecting a certain degree of hypesthesia). The deepest levelof hypesthesia is when a rabbit does not blink when probed with theshortest filament length (e.g., 0.5 cm). Zero degree of hypesthesia (noblockage of pain/no numbing) is when a rabbit blinks when probed withthe longest filament length (e.g., 6 cm). The filament length can beconverted into a pressure (g/mm²) such that 6 cm filament produces 0.4g/mm² pressure (the lowest pressure), while 0.5 cm filament produces15.9 g/mm² pressure (the highest pressure). Therefore, the recordedpressure is the pressure that corresponds to the shortest filamentlength where blink response is not present.

Referring to FIG. 6, the hypesthetic effect (degree of corneal numbingmeasured using tactile stimulation as described herein) was measured inrabbits over time following administration to the eye, with the corneaexposed, of an injected cold slurry (ECT-1719, 3 rabbits in this group,shown with a diamond), a topically applied cold slurry (ECT-4143, 3rabbits in this group, shown with a triangle), and a topically appliedslurry at room temperature (ECT-4143, 1 rabbit in this group, shown witha square). In FIG. 6, the degree of hypesthesia is shown as a percentageof the recorded pressure (i.e., based on the shortest monofilament inwhich there is a lack of blink response) relative to the highestpossible pressure (i.e., shortest monofilament used of 0.5 cmcorresponding to 15.9 g/mm² pressure). The degree of hypesthesia isshown for days 1, 7, 14, and 28 following cold slurry administration.For the injected cold slurry (ECT-1719, shown with a diamond), thehypesthetic effect at day 1 was about 20% and continued to taper offreaching baseline levels by day 14 (error bar overlaps with 0%). For thetopically applied cold slurry (ECT-4143 applied at 18° C., shown with atriangle), the hypesthetic effect at day 1 was 100% (maximal cornealnumbing that could be measured), which then continued to drop, taperingoff at day 28 during which the pain response returned to baseline levels(error bar overlaps with 0%). For the topically applied slurry at roomtemperature (ECT-4143, shown with a square), no hypesthetic effect couldbe observed at any time point following treatment. FIG. 6 thereforedemonstrates an unexpectedly strong hypesthetic effect for topicallyapplied cold slurry which results in long-lasting hypesthesia (almost 1month). Injected cold slurry results in moderate hypesthesia which alsolasted longer than expected (e.g., between about 1 week and 2 weeks).Importantly, for both topical and injection methods, cold slurrytreatment produced long-lasting hypesthesia which normalized back tobaseline levels without causing any permanent numbing effect.

FIG. 7 shows hypesthetic effect in rabbits over time followingadministration to the eye of a topically applied cold slurry (6 rabbits,EC: T-4143) similar to FIG. 6, except with the cornea not exposed(protected with a contact lens). The hypesthetic effect was measured inthe same way as described above with respect to FIG. 6. The hypestheticeffect is shown for days 1, 7, 14, 21, and 28 following treatment withthe topically applied cold slurry. The hypesthetic effect was at about50% at day 1 and tapered off slowly reaching baseline levels by day 21(error bar overlaps with 0%) FIG. 7 therefore demonstrates anunexpectedly moderate-to-strong hypesthetic effect for topically appliedcold slurry (with corneal protection) which results in long-lastinghypesthesia (about 3 weeks) without causing permanent corneal numbing orany corneal damage.

Referring to FIG. 8, representative images of rabbit corneas usingfluorescein staining demonstrate corneal healing over time followingintentional 8 mm corneal abrasion applied to both the control group(FIG. 8A) and following topical application of cold slurry in thetreatment group (FIG. 8B) with protection applied to the corneas andeyelids. The progress of the injury is determined by measuring the sizeof the injury over time. As can be seen in FIG. 8A, corneal healing inthe control group (3 rabbits) occurred at an average healing rate of1.31 mm²/hour in the first 24 hours following corneal abrasion and 0.62mm²/hour between 24 hours and 60 hours following corneal abrasion.Unexpectedly, as shown in FIG. 8B, corneal healing was not impairedcompared to the control group for rabbits that received topicallyapplied cold slurry (ECT-4143). In this group (3 rabbits), cornealhealing following cold slurry treatment occurred at an average healingrate of 1.09 mm²/hour in the first 24 hours following cold slurrytreatment and 0.63 mm²/hour between 24 hours and 60 hours following coldslurry treatment.

Referring to FIG. 9, a graph shows the hypesthetic effect in 6 rabbitsover time after a combination treatment in which a cold slurry wastopically applied first and then injected. In 3 rabbits (shown with adiamond, square, and triangle), the topically applied cold slurry wasECT-1719 which does not contain liposomes which was followed by aninjection of the same cold slurry formulation (ECT-1719). In the other 3rabbits (shown with an “X”, star, and circle), the topically appliedcold slurry was again ECT-1719 (which does not contain liposomes) whichwas followed by an injection of cold slurry that contains liposomes(ECT-4143). The hypesthetic effect is shown as the greatest pressure towhich rabbits did not produce a blink (as described herein withreference to FIGS. 6 and 7). As shown in FIG. 9, the hypesthetic effectcontinued to increase following treatment, likely peaking somewherebetween days 4 and 11, irrespective of combination treatment (liposomalor non-liposomal injection). The hypesthetic effect tapered offreturning to baseline levels at about day 17. Surprisingly, a second,less pronounced period of hypesthesia spontaneously occurred at aroundday 22, tapering back to baseline levels by day 26.

The data described herein support cold slurry (topical and injection) asa long term and safe conical numbing treatment which produceshypesthesia without permanent corneal numbering or damage.

Without being bound by any theory, the basic premise is that applicationof the cold slurry halts the signaling of painful stimuli by causingdegeneration of the myelin sheath over the nerves. Myelin is a fatty,lipid-rich substance that allows electrical stimuli to travel down thenerve axon in a quick and efficient manner. With the administration ofcold slurry over both the free nerve endings and myelinated portion ofthe nerves, the cold temperature will freeze or crystallize the lipidcomponent of fat cells, inducing apoptosis, and degenerating the myelinsheath, a process known as Wallerian degeneration. This process willsignificantly reduce ciliary nerves from conducting painful stimuli fromthe cornea to the brain stem. Due to the sheer volume of distal nerveendings on the ocular surface, not all peripheral nerves are affected,therefore not all sensation from the ocular surface is eliminated, thusinducing relative hypesthesia instead of complete anesthesia.Furthermore, the effect regresses after approximately 4-8 weeks at whichtime ocular sensation is fully restored. Other options that induceWallerian degeneration include radiofrequency ablation andcryoneurolysis (freezing at temperatures approaching −80° C.), but theseprocedures pose the risk of damaging surrounding tissue and structures.Furthermore, an inactive vehicle containing ice crystals will not harmother components of the eye, making it a reasonable application fortreating nerves that lead to ocular surface pain. This approachpreserves sight and normal function of the ocular surface.

Without being bound by a specific theory, injection into thesubconjunctival space surrounding the corneal limbus distributes thecold slurry around the free nerve endings of the ciliary nerves. Thereare two main ciliary nerves that have free nerve endings branching intothe corneas of each eye. Each ciliary nerve is myelinated along itsaxon, which is located downstream from the free nerve endings in thecornea. The injection of the cold slurry spreads downstream to where theaxon of the ciliary nerve is myelinated. As the cold slurry spreads tothe axons of the ciliary nerves, it causes crystallization and apoptosisof the myelin sheath, thus demyelinating the ciliary nerves. Thedemyelination prevents the nerves from transmitting pain signals to thebrain. Alternatively, the cold slurry can cause Wallerian degenerationof the nerve and similarly prevent pain signals from being transmittedto the brain.

The topically applied and/or injected cold slurry is advantageous overother methods of administration because it does not damage the surfaceof the cornea.

The systems and methods disclosed herein are not to be limited in scopeto the specific embodiments described herein. Indeed, variousmodifications of the devices, systems, and methods in addition to thosedescribed will become apparent to those of skill in the art from theforegoing description.

Example 1—In Vivo Testing of Cold Slurry Treatment for Corneal Numbing

The results of the studies described in this Example can be seen inFIGS. 5-9. Preclinical testing with animal studies was performed todetermine the efficacy of the therapy including investigating the bestmeans to deliver the therapy, the duration of the therapy's effect, andany potential side effects. For ophthalmic investigations, the NewZealand white rabbits are the ideal model because their cornea andcorneal innervation system are very similar to humans, and they are astandard accepted model for corneal studies in the literature.

Procedural Preparation

Animals were given a pre-anesthetic (rabbits Xylazine 1.1 mg/kg IMBuprenorphine HCL 0.01-0.05 mg/kg IM) and a pre-surgical antibiotic(Cefazolin 25-50 mg/kg IM). Animals were then anesthetized (rabbitsKetamine 33 mg/kg IM). Animals were placed on heating pad and the vitalsmonitored. Two drops of Proparacaine HCL 0.5% and 5% phenylephrine/0.5%tropicamide (dilating drops) were administered to the eye to be studied.Animals were put on inhalation anesthesia (isoflurane at 1.5-2%concentration) with an O₂ supplement.

Study Procedure

Animals were prepped and draped in the usual sterile fashion includingthe instillation of povidone-iodide drops onto the ocular surface. Aspeculum was placed, and a topical or subconjunctival injection of theslurry is performed.

Injection Administration

For evaluating the hypesthetic potential of ECT-1719, approximately 0.7mL of cold slurry were injected around the corneal limbus into thesubconjunctival space. The injected cold slurry distributes evenly, 360degrees, around the corneal limbus due in part to pressure from thecornea, the force of the injection, and the natural potential spacepresent. The injection procedure is repeated every 120 seconds for atotal of 10 minutes.

Control animals received treatment with sterile saline (control) ortreatment with a vehicle control (uncooled slurry). At the conclusion ofthe procedure, the eye was carefully inspected, the speculum removed,the drapes removed, and the eye washed with sterile saline. There was anadditional control group that has conventional anesthetic drops appliedto the cornea. All surgery is on the left eye only (for controlpurposes) and lasted about 10 minutes.

The surgical procedure described above is commonly performed in humanswith injection of a variety of different agents depending on thecondition (e.g. steroids, antibiotics, etc.).

Topical Administration

For evaluating the hypesthetic potential of ECT-4143, the slurry wasapplied topically to the ocular surface, posterior to the cornea limbus.The cornea was protected with a contact lens and the eyelids with aplastic speculum. Approximately 2-3 mL of cold slurry were appliedtopically every approximately 90 seconds, with approximately 2-3 ml perapplication, until a total treatment time of 10 minutes was reached.).At the conclusion of the procedure, the eye was carefully inspected, thespeculum removed, the drapes removed, and the eye washed with sterilesaline.

Post-Procedures for a Survival Animal

A Neomycin/Polymyxin/Bacitracin ophthalmic ointment was applied to theoperative eye, as well as several drops of Prednisone Acetatepostoperatively. Animal were removed from a surgical table and placed ona heating pad. Animals had their vitals monitored (e.g., heart rate,breathing, SPO₂) while waiting for recovery. Animals continued to bemonitored until regaining muscle control. Animals were returned to theirrespective cage of origin.

Post-Surgery Animal Monitoring

Animals receive a comprehensive eye exam one day after surgery and thenweekly which included a measurement of corneal sensation. A measurementof intraocular pressure was taken as well, as a beneficial lowering ofintraocular pressure may be observed in animals that have undergone thistherapy. Furthermore, a slit lamp exam with fluorescein staining anddilated fundus exam (I.e., eyes are dilated with 5% phenylephrine, 0.5%tropicamide) was performed. Animals were placed in restrictive cages fora few seconds while eye drops were instilled.

Effect of Administration

The effect of the administration of the cold slurry were examined usinga number of techniques.

The cold slurry's numbing effect was tested using an esthesiometer. Afilament was extended from the device that had a certain stiffness. Theanimals treated with cold slurry were able to tolerate more force fromthe esthesiometer than animals in the control groups. This wasdemonstrated by whether an animal flinches when poked in the eye withthe esthesiometer filament. The test was administered multiple timesover the course of the study to determine the length of the numbingeffect.

The impact of the cold slurry on the eye's ability to heal was alsoexamined. An epithelial defect on the cornea was created with a trephineand corneal brush. The injury was verified with a fluorescein stain andphotodocumented. The progress of the injury was measured usingfluorescein staining and measuring the size of the injury. The coldslurry did not impact the eye's ability to heal.

What is claimed is:
 1. A method of alleviating symptoms of ocularsurface discomfort, the method comprising: topically applying a coldslurry adjacent to a corneal limbus of an eye of a patient, wherein thecold slurry comprises water and a freezing point depressant, wherein thetopical application of the cold slurry is configured to cause a degreeof numbing of a cornea of the eye for a period of time, said period oftime being more than about two days, and wherein an ocular sensation ofthe eye is restored following the period of time.
 2. The method of claim1, wherein the cold slurry is applied posterior to the corneal limbus.3. The method of claim 1, wherein the period of time is more than about7 days without topically applying the cold slurry an additional time onany day following the first day of topical application.
 4. The method ofclaim 3, wherein the freezing point depressant is glycerol.
 5. Themethod of claim 1, wherein the ocular sensation of the eye is restoredafter about 21 days following the topical application of the coldslurry.
 6. The method of claim 1, wherein a sclera of the eye of thepatient is cooled to a temperature of between about −6° C. and about 4°C. during the topical application of the cold slurry.
 7. The method ofclaim 1, wherein the cold slurry is topically applied for between about5 minutes and about 15 minutes.
 8. The method of claim 7, wherein anadditional amount of the cold slurry is topically re-applied at aboutevery 90 seconds.
 9. The method of claim 1, further comprising placing acontact lens on the eye of the patient prior to topically applying thecold slurry.
 10. The method of claim 1, wherein the cold slurry isconfigured to be a paste consistency.
 11. A method of alleviatingsymptoms of ocular surface discomfort, the method comprising: placing aprotective covering over a cornea of an eye of a patient; and topicallyapplying a cold slurry to a bulbar conjunctiva of the eye of thepatient, wherein the topical application of the cold slurry causes aprolonged reduction of pain of the eye of the patient, said prolongationbeing a period of time of more than about two days, and wherein apartial sensation of the cornea of the eye of the patient is maintainedduring the prolonged reduction of pain.
 12. The method of claim 11,wherein the cold slurry is applied posterior to the corneal limbus. 13.The method of claim 11, wherein the cold slurry is applied over theprotective covering.
 14. The method of claim 11, wherein the prolongedreduction of pain lasts more than about 7 days without topicallyapplying the cold slurry an additional time on any day following thefirst day of topical application.
 15. The method of claim 14, whereinthe prolonged reduction of pain lasts more than about 14 days withouttopically applying the cold slurry an additional time on any dayfollowing the first day of topical application.
 16. The method of claim11, wherein the symptoms are due to dry eye syndrome or cornealsomatosensory dysfunction.
 17. The method of claim 11, wherein a scleraof the eye of the patient is cooled to a temperature of between about−6° C. and about 4° C. during the topical application of the coldslurry.
 18. The method of claim 11, wherein the protective covering is acontact lens, and wherein the contact lens prevents the cornea of theeye from freezing.
 19. A method of alleviating symptoms of ocularsurface discomfort, the method comprising: administering a cold slurryto an eye of a patient, wherein the cold slurry comprises water and apercentage of ice particles, wherein the administration of the coldslurry causes a prolonged hypesthesia of the eye, said prolongationbeing a period of time of more than about two days, wherein an ocularsensation of the eye is restored following the prolonged hypesthesia,and wherein the administration of the cold slurry does not causepermanent damage to a cornea of the eye.
 20. The method of claim 19,further comprising treating a condition selected from the groupconsisting of dry eye syndrome, chronic eye pain, post-operative pain,post-photorefractive keratectomy pain, post-LASIK pain, post-cataractsurgery pain, and post open globe injury repair pain, post-cornealinjury, corneal somatosensory dysfunction, allodynia, and pain fromacute injury.
 21. The method of claim 19, wherein the cold slurry isadministered via injection.
 22. The method of claim 19, wherein the coldslurry is injected into a subconjunctival space.
 23. The method of claim19, wherein the cold slurry is administered via topical application. 24.The method of claim 19, wherein the percentage of ice particles isbetween about 20% and 40%.
 25. The method of claim 19, wherein thetemperature of the cold slurry is between about −20° C. and about −5° C.26. A method of alleviating symptoms of ocular surface discomfort, themethod comprising: topically applying a cold slurry on or proximal to anocular surface of an eye of a patient, wherein the topical applicationof the cold slurry causes a prolonged hypesthesia of a cornea of theeye, said prolongation being a period of time of more than about twodays, wherein an ocular sensation of the eye is restored following theprolonged hypesthesia, and wherein the topical application of the coldslurry does not cause permanent damage to the cornea of the eye.
 27. Themethod of claim 26, wherein the cold slurry is applied proximal to thecorneal limbus.
 28. The method of claim 26, wherein the ocular sensationof the eye is restored by about 30 days following the topicalapplication of the cold slurry.
 29. The method of claim 26, wherein thecold slurry is topically applied for between about 5 minutes and about15 minutes.
 30. The method of claim 26, further comprising placing acontact lens on the eye of the patient prior to topically applying thecold slurry, and wherein the contact lens prevents the cornea of the eyefrom freezing.